Applications

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application

Abreviews

Notes

IP

Use at an assay dependent concentration.

WB

1/100. Detects a band of approximately 100 kDa. In pig brain extract detects a doublet of approximately 100 kDa representing the two isoforms of alpha-adaptin.

ICC/IF

1/100.

ELISA

Use at an assay dependent concentration.

IHC-P

1/20.

Flow Cyt

1/100.

ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.

Target

Relevance

Clathrin mediated endocytosis is the pathway by which many receptors for nutrients and hormones are internalized to be recycled or down regulated. During formation of clathrin coated membranes, clathrin co assembles with heterotetrameric molecules known as assembly polypeptides (APs) or adaptors which form a layer of protein coat between the clathrin lattice and the membrane. There are two characterized adaptors AP1 and AP2. AP1 is associated with clathrin coated vesicles at the trans Golgi network and AP2 is associated with the endocytic clathrin coated vesicles at the plasma membrane and has been shown to specifically interact with Shc and EGF receptor. AP2 is composed of four subunits, two separate 100 kDa gene products with similar domain structures (alpha and beta adaptin) and a 50 and 17 kDa subunit. There are two alpha adaptin genes, alpha A and alpha C which have a tissue specific pattern of expression.

Images

Immunohistochemistry was performed on normal biopsies of deparaffinized Human heart tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer and microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:20 with a Mouse monoclonal antibody recognizing alpha Adaptin (ab2807) or without primary antibody (negative control) overnight at 4�C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Overlay histogram showing HepG2 cells stained with ab2807 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab2807, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HepG2 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.